Invariance of exocytotic events detected by amperometry as a function of the carbon fiber microelectrode diameter.

Etched carbon fiber microelectrodes of different radii have been used for amperometric measurements of single exocytotic events occurring at adrenal chromaffin cells. Frequency, kinetic, and quantitative information on exocytosis provided by amperometric spikes were analyzed as a function of the surface area of the microelectrodes. Interestingly, the percentage of spikes with foot (as well as their own characteristics), a category revealing the existence of sufficient long-lasting fusion pores, was found to be constant whatever the microelectrode diameter was, whereas the probability of overlapping spikes decreased with the electrode size. This confirmed that the prespike foot could not feature accidental superimposition of separated events occurring at different places. Moreover, the features of amperometric spikes investigated here (charge, intensity and kinetics) were found constant for all microelectrode diameters. This demonstrated that the electrochemical measurement does not introduce significant bias onto the kinetics and thermodynamics of release during individual exocytotic events. All in all, this work evidences that information on exocytosis amperometrically recorded with the usual 7 microm diameter carbon fiber electrodes is biologically relevant, although the frequent overlap between spikes requires a censorship of the data during the analytical treatment.

Concerted activities of nitric oxide synthases and NADPH oxidases in PLB-985 cells.

Oxidative stress is a metabolic situation used by immune cells to provide protection against infection. Under activation by threatening elements, phagocytes produce chemically toxic molecules, namely the reactive oxygen species (ROS) and reactive nitrogen species (RNS). This mechanism involves two types of enzymes: NAPDH oxidases (NOX) and NO synthases (NOS), which activities are versatile and not fully understood yet. In this regard, we studied in real-time the release of bursts of ROS and RNS by single PLB-985 cells, originating from a myeloid cell line prone to differentiate into neutrophil or monocyte-like phagocytes. A selective electrochemical detection of each ROS or RNS was conducted at platinized carbon fiber microelectrodes positioned at micrometric distances from single cells. Our results show (1) the existence of a NO synthase activity in PLB-985 cells and (2) the ability of NO synthases to provide a NOX activity in cells where NADPH oxidase (NOX2) is knocked out.

Relationship between amperometric pre-spike feet and secretion granule composition in chromaffin cells: an overview.

Amperometry is a simple and powerful technique to study exocytosis at the single cell level. By positioning and polarizing (at an appropriate potential at which the molecules released by the cell can be oxidized) a carbon fiber microelectrode at the top of the cell, each exocytotic event is detected as an amperometric spike. More particularly, a portion of these spikes has previously been shown to present a foot, i.e. a small pedestal of current that precedes the spike itself. Among the important number of works dealing with the monitoring of exocytosis by amperometry under different conditions, only a few studies focus on amperometric spikes with a foot. In this work, by coupling our previous and recent experiments on chromaffin cells (that release catecholamines after stimulation) with literature data, we bring more light on what an amperometric foot and particularly its features, represents.

Comparison of apex and bottom secretion efficiency at chromaffin cells as measured by amperometry.

In chromaffin cells, the exocytosis of neuromediators involves the fusion between a secretory vesicle and the cell membrane. Many techniques based on electrophysiology, electrochemistry and fluorescence microscopy allow the study of such a complex process at active zones of single immobilized cells. These techniques can provide an effective analysis either at the apex, either at the base of the cell adhering onto a substrate. For instance, patch-clamp (electrophysiology) and amperometry (electrochemistry) deal with detection at the exposed top of the cell, whereas evanescent field microscopy concerns mainly its bottom, i.e., the zone on which the cell rests onto the surface. However, in chromaffin cells, comparison between the two sets of methods remains to be established and whether apex fusion events are comparable or not to those observed at the base of the cell is an open question. In this work, we compare both active zones upon using the same measurement method, viz., by performing electrochemical detection at these both poles (top and bottom) of bovine chromaffin cells. This is performed upon using carbon fiber microelectrodes (apical analysis) and planar ITO transparent (basal analysis) electrodes, respectively. Our results indicate that the processes monitored at each pole differ though the same technique is used.

Vesicular exocytosis under hypotonic conditions shows two distinct populations of dense core vesicles in bovine chromaffin cells.

Several previous reports have discussed the effects of external osmolarity on vesicular exocytotic processes. However, few of these studies considered hypotonic conditions on chromaffin cells. Herein, the exocytosis of catecholamines by chromaffin cells was investigated in a medium of low osmolarity (200 mOsm) by amperometry at carbon fiber microelectrodes. It is observed that the frequency of the exocytotic events is significantly higher under hypotonic conditions than under physiological conditions (315 mOsm). This further confirms that the swelling of the polyelectrolytic matrix (which follows ionic exchanges) contained in dense core vesicles is the energetic driving force of the exocytotic phenomenon, being favored by a lower osmolarity. The mean amount of catecholamines released during secretory events also increases importantly under the hypotonic condition. This may be rationalized by the coexistence of two distinct populations of dense core vesicles with a relative content ratio of 4.7. The larger content population is favored under hypotonic conditions but plays only a side role under isotonic conditions.

Regulation of exocytosis in chromaffin cells by trans-insertion of lysophosphatidylcholine and arachidonic acid into the outer leaflet of the cell membrane.

Vesicular exocytosis is an important complex process in the communication between cells in organisms. It controls the release of chemical and biochemical messengers stored in an emitting cell. In this report, exocytosis is studied amperometrically (at carbon fiber ultramicroelectrodes) at adrenal chromaffin cells, which release catecholamines after appropriate stimulation, while testing the effects due to trans-insertion of two exogenous compounds (lysophosphatidylcholine (LPC) and arachidonic acid (AA)) on the kinetics of exocytotic events. Amperometric analyses showed that, under the present conditions (short incubation times and micromolar LPC or AA solutions), LPC favors catecholamine release (rate, event frequency, charge released) while AA disfavors the exocytotic processes. The observed kinetic features are rationalized quantitatively by considering a stalk model, for the fusion pore formation, and the physical constraints applied to the cell membrane by the presence of small fractions of LPC and AA diluted in its external leaflet (trans-insertion). We also observed that the detected amount of neurotransmitters in the presence of LPC was larger than under control conditions, while the opposite trend is observed with AA.